In normal airway epithelial cells, chloride channels can be activated by agents which raise intracellular c-AMP. CF airway epithelial cells have been shown to contain chloride channels which cannot be activated by a normal increase in intracellular c-AMP. The defect in CF may involve some step in an activation sequence distal to the generation of c-AMP. Alternatively, CF cells could cause Cl-channel activity to be tonically inhibited. We propose to isolate and identify substances in the cytosol or associated with cell membranes which control the activity of single chloride channels in CF and non-CF airway epithelial cells. The specific aims of this proposal are: (1) Assay cytoplasm and membrane fractions derived from CF and non-CF cells for the ability to activate or deactivate chloride channels in the apical cell membrane of CF and non-CF cells and compare these effects to the effects of purified protein kinases or phosphatases. (2) Identify and isolate c-AMP dependent protein kinase activity, phosphatase activity, and phosphoproteins in cytoplasm and membrane fractions derived from CF and non-CF cells and assay for ability to alter ion channel function. The results from the proposed experiments should reveal if CF cells produce an inhibitor of ion channel activation, or if CF cells are missing a portion of the normal activation sequence of Cl channels found in normal cells.